BMMs seeded in 12-well plates were infected with Schu S4 at a MOI of either 200 (0, 1, 2 and 4 h time points), 50 (8, 12 h time points) or 25 (16 and 24 h time points). The use of different MOIs in this experimental design was required to recover sufficient amount of bacterial RNA at all time points analyzed but did not affect the timing of the Francisella intracellular cycle, therefore generating biologically comparable samples. Time zero samples were generated by adding bacteria (MOI 200; 108/well) directly to BMMs that had been washed with PBS, followed by the immediate addition of lysis buffer, as described below. Uninfected controls were generated at both 0 and 24h post infection.
Growth protocol
Bone marrow cells were isolated from femurs of 6-10 week-old, C57BL/6J female mice (Jackson Laboratories, Bar Harbor, ME, USA) and differentiated into macrophages for 5 days at 37°C and 7% CO2, in 1g/L glucose Dulbecco's Modified Eagle Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 10% L-conditioned medium {de Chastellier, 1993 #28}, and 2 mM L-glutamine in non tissue culture-treated Petri dishes. After 5 days, loosely adherent BMMs were washed with PBS, harvested by incubation in chilled cation-free PBS on ice for 10 min, resuspended in complete medium and replated in either 6-, 12- or 24-well cell culturetreated plates at a density of 1x106, 5x105 or 1x105 macrophages/well, respectively. BMMs were further incubated at 37°C under 7% CO2 atmosphere for 48 h, with replenishing complete medium 24 h before infection. For infections, bacterial suspensions were diluted in complete medium and 1.5, 1 or 0.5 ml was added to chilled BMMs at an appropriate multiplicity of infection (MOI) of 50, unless stated otherwise. Bacteria were centrifuged onto macrophages at 400 x g for 10 min at 4°C, and infected BMMs incubated for 20 min at 37°C under 7% CO2 atmosphere including an initial, rapid warm up in a 37°C water bath to synchronize bacterial uptake. Infected BMMs were 23 then washed 5 times with DMEM to remove extracellular bacteria, incubated for an additional 60 min in complete medium containing 100 μg/ml gentamicin to kill extracellular bacteria and thereafter in gentamicin-free medium until processing.
Extracted molecule
total RNA
Extraction protocol
At each time point, samples were washed 3 times with sterile PBS to remove residual extracellular bacteria, and both BMMs and bacteria were lysed directly in the wells by the addition of 1 ml TRIzol (Invitrogen, Carlsbad, CA) and pipetting 10 times. Samples were transferred to microtubes, 200 ìl chloroform were added, vials were vortexed, and centrifuged at 16,000 x g for 15 min. The RNA-containing aqueous phase was collected from each sample and passed through a Qiashredder column (Qiagen, Valencia, CA) at 21,000 x g for 2 min.
Label
biotin
Label protocol
RNA amplification step was introduced as follows: all RNA samples that were generated from F. tularensis SchuS4-infected and noninfected BMMs were randomized on a single 96-well plate and the MessageAmp II-Bacteria kit (Ambion, Foster City, CA) was used to amplify 0.5 to 1 μg of mixture of F. tularensis and BMM RNAs according to manufacturer's instructions. Briefly, RNAs were concentrated to 5 μl, denatured for 10 min at 70°C, polyadenylated using E.coli polyA polymerase for 15 min at 37°C, and reverse transcribed for 2 h at 42°C using ArrayScript and polydT primer with a T7 tail. Second strand synthesis was performed immediately after first strand cDNA synthesis for 2 h at 16°C. Purified cDNA was mixed with 75 nmol of biotin-16-UTP (Roche Applied Science, Mannheim, Germany) and concentrated to 18 μl. Biotin-labeled complementary pathogen-host RNA was amplified at 37°C in an overnight 40 μl in vitro transcription reaction. The amount of target consisting of macrophage and F. tularensis cRNAs was determined by Absorbance measurements at 260 nm. The quality and the size of the amplified targets were determined on a Agilent 2100 Bioanalyzer using the Pico analysis kit (Agilent Technologies, Palo Alto, CA). The amount of F. tularensis cRNA was estimated by quantitative RT-PCR of the FTT0243 locus, as described below, using a standard curve method (Applied Biosystems, Foster City, CA). F.tularensis RNA extracted from a bacterial suspension in TSB-C was used for the RNA standard curve.
Hybridization protocol
~1ug label and fragmented applied for 16hrs 40OC
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 7GPlus.
Description
Gene expression data from_time point zero rep 1
Data processing
The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Ft genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
